Plants offer enormous potential for production of recombinant forms of therapeutics and diagnostics for humans and animal applications, for example, human blood proteins, antibodies, vaccines, and the like. Plants have the ability to perform complex post-translational modifications and are intrinsically safe since they do not propagate mammalian viruses or pathogens. Plants also offer a lower cost alternative and are easier to scale-up compared to traditional mammalian cell culture production methods (Goldstein and Thomas, 2004; Ma et al., 2003). Thus, the potential for plant expression systems to serve as large scale production methods of therapeutic and diagnostic proteins, including the production of enzymes is compelling. Transient production approaches utilizing agroinfiltration or plant viral infection of plant tissue are particularly promising for short production timelines due to the relatively short time needed to go from gene to product, and the ability to use available agricultural biomass for rapid, large scale production (Fischer et al., 1999). Despite the ease and short turnaround time for transient production, use of recombinant plant viruses has been hindered by various shortcomings, including low infection efficiency, the risks associated with the release of the competent recombinant virus vectors into the environment (Manske and Schiemann, 2005), limitations on the size of the transgene, and transgene instability (Rabindran and Dawson, 2001; Shivprasad et al., 1999).
Agrobacterium-mediated infection to deliver viral amplicons was first reported in 1993 (Turpen et al., 1993). Gleba et al. (2005) described a plant virus based expression technology, termed “magnifection”, wherein whole plants are vacuum infiltrated with A. tumefaciens containing a binary vector capable of expressing recombinant viral RNA replicons able to move from cell-to-cell, multiply, and express amplified levels of transgenes carried by the viral replicon. Gils et al. have shown high level production of correctly processed, biologically active human growth hormone using transient agroinfiltration of N. benthamiana leaves with multiple constructs that allow the in planta assembly of TMV viral replicons (Gils et al., 2005). Production of heterologous proteins utilizing constitutively-expressed viral amplicons has also been investigated in stably transformed plants, however, low recombinant viral levels, and low product titers resulting from post-transcriptional gene silencing (PTGS) have been continuously problematic (Angell and Baulcombe, 1997; Mori et al., 1993). In order to address this issue, Mori et al. (2001) constructed a chemically inducible Brome mosaic virus (BMV)-based amplicon utilizing the dexamethasone (DEX) glucocorticoid-inducible transcription system (Aoyama and Chua, 1997) for production of human gamma interferon in transgenic Nicotiana benthamiana. A similar approach was used for protein expression using a plant DNA virus amplicon (Zhang and Mason, 2006).
However, low recombinant viral levels, and low product titers resulting from post-transcriptional gene silencing continue to be a problem. Thus, with increasing threats of global pandemics and bioterrorism, there is a critical need for new bio-manufacturing technologies that allow rapid, large scale and cost-effective production of diagnostic, therapeutic and prophylactic compounds that can be used for detection, treatment or vaccination following such an event. The invention addresses this need.
There is also an increasing need for renewable and more environmentally friendly alternatives and supplements to fossil fuels and the efficient, low cost and scalable production of enzymes that are involved in the biosynthesis of such fuels. While bioethanol and biodiesel are rapidly expanding as renewable, more environmentally friendly alternatives to fossil fuel derived gasoline and diesel, new technologies that allow, for example, energy efficient and cost efficient degradation of lignocellosic biomass would represent a major breakthrough. Although there have been advances in improving the activity of enzymes involved in the degradation of lignocellulosic biomass, design of engineered cellulase mixtures, development of more productive host strains, and utilization of inexpensive medium components, the high cost of producing, recovering and formulating cellulase enzymes using traditional fungal or bacterial fermentation continues to impact the economics of ethanol production from lignocellulosic biomass. Microbial fermentations require large energy inputs due to agitation, aeration, temperature control and in some cases cell disruption and enzyme recovery, as well as high capital equipment costs for fermentors and downstream processing unit operations. The application of cellulases often requires pretreatment of lignocellulosic biomass to facilitate accessibility of the enzymes to the complex substrate, again requiring additional energy inputs and/or costly treatment of acid/base wastes. Thus, a new approach for controlled, in planta production of enzymes involved in lignocellulosic degradation using chemically inducible, transient, high level expression of cellulase enzymes produced in plants is highly desirable and the invention addresses this need.